Self-assembling peptide surfaces for cell patterning and interactions

ABSTRACT

This invention describes self assembled monolayers (SAMs) manufactured by imprinting reactive peptides onto solid supports. The invention further relates to methods of preparing and using these improved SAMs.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/317,838, filed Dec. 11, 2002, which is a continuation of U.S.application Ser. No. 10/071,500, filed Feb. 8, 2002, which is acontinuation of U.S. application Ser. No. 08/882,415, filed Jun. 25,1997. The entire teachings of the above applications are incorporatedherein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant (Grant No.DAAH04-94-G-0407) from the Army Research Office. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Organic surfaces have been employed in numerous methods and systems,including as substrates for ELISA, cell and tissue culture.Self-assembled monolayers (SAMs) are a class of organic surfacesmanufactured by imprinting a monolayer of organic compounds withreactive moieties onto a solid support under conditions wherein thecompounds react with and bind to the solid support in a single orderedand patterned layer. See, Lopez, et al., “Convenient Methods forPatterning the Adhesion of Mammalian Cells to Surfaces UsingSelf-Assembled Monolayers of Alkanethiolates on Gold,” J. Am. Chem.Soc., 115(13):5877-5878 (1993) and Mrksich and Whitesides, “UsingSelf-Assembled Monolayers to Understand the Interactions of Man-MadeSurfaces with Proteins and Cells”, Annu. Rev. Biophys. Biomol. Struct.,25:55-78 (1996). Molecular self-assembly is the spontaneous associationof molecules under equilibrium conditions into stable, structurallywell-defined order joined by non-covalent bonds. SAMs manufactured todate have linked chemical moieties to solid surfaces through long chainalkyl linkages. Examples of organic compounds which have been patternedon a solid support include alkanethiolates and alkylsiloxanes. The SAMsare manufactured employing a process termed “microcontact printing.”

It has been suggested that SAMs can be used to pattern cells on asurface by presenting chemical moieties which bind to the cells on thesolid surface. Mrksich and Whitesides, above. However, these molecules,and the resulting SAMs, can be difficult and/or expensive tomanufacture. Thus, improvements and cost reductions in the manufactureof SAMs are desirable and are necessary.

SUMMARY OF THE INVENTION

This invention is based upon the discovery that improved SAMs can bemanufactured by imprinting reactive self assembling peptides onto solidsupports. The SAMs are characterized by ease of manufacture andpurification. They are versatile in their ability to readily provide alarge variety of chemical reactive moieties, or “presenting groups”, toselected targets. For example, the SAM's of the present invention can bereadily designed to present ligands to cellular receptors, cell adhesionmotifs, antibodies or antigen-binding fragments thereof to cell surfaceproteins. This preferred class of SAMs can be used to bind a target,e.g. a selected cell or cells, to a predetermined locus on the solidsupport.

Thus, the invention relates to a composition of matter comprising asolid support and a self-assembled monolayer of linear peptides whereinsaid peptides bound directly to said solid support through a terminalamino acid in a predetermined pattern. Preferably, the peptides comprisea terminal reactive group, a central linker and a presenting group. Theinvention also relates to the uses and applications of the SAMsdescribed herein, as will be described in more detail below.

The invention further relates to a method for manufacturing an SAM, or acomposition of matter comprising a solid support and a self-assembledmonolayer of linear peptides wherein said peptides bound directly tosaid solid support through a terminal amino acid in a predeterminedpattern, comprising microcontact printing the reactive peptides onto thesolid support and maintaining the peptides under conditions suitable forbinding.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings. The drawings are not necessarily to scale,emphasis instead being placed upon illustrating the principals of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate methods of microcontact printing reactivepeptides to a solid support in a predetermined pattern.

FIG. 3 illustrates patterns which may be selected, for example, in SAMsfor immobilizing cells.

DETAILED DESCRIPTION OF THE INVENTION

As set forth above, the invention relates to improved SAMs comprising apredetermined pattern of peptides on a solid support. Preferred peptidesof the invention can be characterized by three regions bound to eachother through an amino acid or via peptide binding, the “terminalreactive group”, the “central linker” and the “presenting group.”

Upon binding the peptides to the solid support, the peptides arepreferably highly ordered and preferably possess a consistent linear andparallel configuration with each other. Generally, the peptides, or thecentral linker thereof, are fully extended beta strands in configurationunder the conditions of use.

Although in some embodiments, it may be desirable to present a ligand orother molecule which possesses a tertiary structure, generally, thepeptides are linear (e.g., free or substantially free of branching ortertiary structure). “Substantially free” of branching or tertiarystructure is intended to include minor amounts of branching and peptideinteractions which do not significantly interfere with the free movementor function of the presenting group. The actual degree of branching andpeptide interactions which can be tolerated without deleteriouslyeffecting the quality of the product will be function of the overalllength of the peptide, the branched peptides, the nature of the aminoacids in each and their ability or tendency to interact with each othercan generally be determined by routine screening or computer modeling.For example, peptides “substantially free” of branching may include apeptide composition wherein less than about 5% of the peptides arecharacterized by one or more branches.

While the length of the peptide is not critical to the invention, thepeptide is preferably small to moderate in length. Thus, the centrallinker of the peptide can preferably be between about 2 to about 50naturally occurring or non-naturally occurring amino acids in length arepreferred, more preferably between about 8 to about 35 amino acids inlength. Certain peptides in excess of 50 may present undesirableinteractions of the peptides, such as a possible tendency of the peptideto fold. Peptide interactions can be predicted by, for example, computermodeling and structural information available at protein data banks at,for example, Brookhaven National Laboratories, N.Y.

Peptides which can be used in the invention can be characterized by areactive moiety which can react and bind to the solid support, the“terminal reactive group”. Typically, the terminal reactive group is anamino acid characterized by a functional group pendant from the sidechain, the amino group or the carboxy group. Thus, the terminal reactivegroup which binds to the solid support can be an amino acid substitutedby a hydroxy, thiol, carboxy, amino, amido, imido or guanidino group.Preferred terminal amino acids, thus, include serine, cysteine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,arginine and histidine. Alternatively, the terminal reactive group canbe a nonnaturally-occurring amino acid characterized by a functionalitywhich can react with the solid support. Examples include beta aminoacids (amino acids wherein the amino and/or carboxy group are notsubstituted on the same alpha carbon, such as beta-alanine) or aminoacids which have been chemically modified, by electrophilicsubstitution, nucleophilic substitution, activation reactions oraddition reactions, for example. See March, “Advanced Chemistry,” ThirdEdition (1985), Chapters 10-16, the contents of which are incorporatedherein by reference.

It is further desirable that the peptide be of sufficient length toprovide a flexible, spatial separation between the solid support (uponreaction with the reactive terminal group) and the opposing reactiveterminus of the peptide (e.g., the presenting group). Thus, the peptidesof the invention preferably comprise a “central linker”, which is apeptide bound to the terminal reactive group and presenting groupthrough peptide or amide bonds. The amino acids employed in the peptideand/or central linker are selected to promote or optimize a beta strandconfiguration at the conditions for use. It is further preferred thatthe amino acids in this portion of the peptide be substantially free oflarge or bulky side chains or bonds which will interfere with theconfiguration (e.g. proline). The amino acids can further be selectedconsidering material strength, permeability and degradation rate of theresulting peptide and SAM. Preferably, the amino acids selected for thecentral section of the peptide are glycine, L-alanine and D-alanine.D-amino acids have advantages in many applications due to theirresistance to L-protease degradation.

The length of the central linker where present, is also generally notcritical to the invention. Preferably, the central linker is betweenabout 2 and about 30 amino acids in length, more preferably betweenabout 2 and about 8 amino acids.

The peptide can also be characterized by a “presenting moiety” whichwill bind to one or more targets. The term “presenting group” is definedherein to include one or more chemical atoms, functional groups, aminoacids or peptides that possess an affinity to or resistance for a targetentity. For example, a presenting group which presents a resistance fora target entity, e.g., a protein or cell, can be poly(ethylene glycol)or another compound which is inert to the target. A presenting groupwhich is resistant to water, as a target molecule, is a hydrophobicgroup, such as a high chain alkyl or hydrophobically blocked amino acid(e.g., an alkyl ester of valine, leucine, isoleucine or phenylalanine).

Generally, one or more peptides employed in the present inventionpossess a presenting group with an affinity for a target, e.g. a targetmolecule. In such embodiments, the presenting group can be specific ornon-specific for the target molecule. For example, where the target is acell, the target molecule can be a cell surface protein. The presentinggroup can be a ligand for that protein, an antibody or anantigen-binding fragment thereof which binds specifically to the cellsurface protein.

The presenting group can be a non-peptide or, preferably, a peptide. Asdiscussed above, the presenting group can be a ligand for or an antibodyor antibody fragment which binds to the target molecule.

Particularly suitable presenting groups are oligopeptides which selfassemble to form a beta sheet under conditions for the desired orselected application. Examples of oligopeptides which self assembleunder these conditions are described in U.S. application Ser. Nos.08/346,849 and 08/784,606, which are incorporated herein by reference intheir entireties. Briefly, these oligopeptides are amphiphilic, havealternating hydrophobic and hydrophilic amino acids and arecomplementary. As will be described in more detail below, particularlypreferred oligopeptides for self assembly are RADX_(n) and EAXX_(n)wherein X is an amino acid and n is an integer between about 2 and about8.

Particularly preferred targets include cells. Examples of cells whichcan be targeted include prokaryotic and eukaryotic cells. The cells canbe mammalian, plant, bacterial, and yeast. Mammalian cells which can betargeted include tumor cells, normal somatic cells and stem cells. Thecells can be fibroblasts, endothelial cells, neuronal cells,hepatocytes, blood cells, smooth muscle cells, and progenitors thereof,for example. Bacterial cells can be gram positive or gram negativebacteria and can include Escherichia coli, Streptococcus,Staphylococcus, as well as many others. Bacterial cells which may bedesirable to target and, thus detect and/or culture, can includepathogens and non-pathogens, e.g., contaminants in a food sample, amammalian tissue sample or serum sample or in a plant tissue sample.Similarly, yeast can be targeted and include, for example, Candida andSaccharomyces.

Cells can preferably be targeted by selecting a presenting group whichwill react with and bind to the cell surface. Generally, this isaccomplished by binding to a cell surface molecule, such as a protein,lipid, or sugar at the surface of the protein. These surface moleculesare included herein as “target molecules.” For example, a targetmolecule can be a cell surface protein and can be specific to the targetor, in this case, cell, or the target molecule can be non-specific.Where the object of the application is to detect the presence of a cellin a sample, e.g., a tumor cell in a sample which can contain normalcells, it is desirable that the target molecule be specific to the tumorcell (e.g., present on tumor cells and absent on the normal cells).These molecules are generally known in the art as tumor markers. Wherethe object of the invention is to detect the presence of bacteria in asample, such as in food, tissue sample, blood sample, or pharmaceutical,it can be desirable to select a target molecule which is present on manytypes of bacteria which are potentially contaminating the sample to betested. In other instances, e.g., where a substantially pure cellculture is being targeted or transferred to the solid support, theselection of specific or non-specific target molecule is immaterial.

Suitable target molecules include tumor markers, cellular receptors,such as CD4 and, CD8. Neuronal cellular receptors include N-CAMs, the L1receptors, NGF receptor, the netrin receptor and others.

Targets can include non-cellular products as well, including viruses(such as retroviruses, influenza viruses, and herpesviruses, forexample), and proteins (such as prostate soluble antigen (PSA),cytokines, cytokine receptors, growth factors, and growth factorrecpetors. Where the target is a virus, the target molecule can be asurface protein as well, such as a cellular receptor implicated in theinfection of cells. A particularly preferred target molecule for HIV is,for example, gp120.

Examples of presenting groups include cellular adhesion motifs, ligandsor binding fragments of ligands for the target molecule (e.g., theligand for gp120 is CD4), antibodies or antigen binding fragments ofantibodies which bind to the target molecule.

A ligand is defined here to include molecules which are the same as orsubstantially the same as the native molecule which binds the targetmolecule. For example, CD4 is a native ligand for the HIV env protein,gp120. Thus, where the target molecule is gp120, the term “ligand” and,thus, the presenting groups include native CD4, a ligand-bindingfragment of CD4 (such as, an extracellular domain), and mutationsthereof which bind to gp120.

The terminal reactive group, central linker and presenting group arepreferably arranged linearly with the central linker bonded directly orindirectly to both the reactive group and the presenting group through,e.g., peptide bonds. Preferably, the peptide has the formula:X—(CH₂)_(n)—CH(NH₂)CO(AA)_(m)-LorX—(CH₂)_(n)—CH(COOH)NH(AA)_(m)-L

wherein X is an inert group, such as H, alkyl, alkoxy, alkylthio ordialkylamine, or is a labile or reactive group, such as a thiol,hydroxy, amino, carboxy, acylhalide, carboxy ester, or halide;

AA is, independently, the same or different, naturally-occurring ornon-naturally-occurring amino acid, and is preferably, glycine,L-alanine or D-alanine;

L is a group which binds specifically or non-specifically to a targetand is preferably a peptide, such as a ligand, an antibody or anantibody fragment;

n is zero or an integer between 1 to about 5;

m is an integer of at least about 2 and, preferably, between about 2 andabout 50, more preferably between about 2 and about 8.

The peptides of the invention can be manufactured by known and industrystadnard peptide synthesis technology. For example, the peptides can besynthesized chemically or recombinantly (e.g. by the expression of arecombinant nucleic acid molecule which encodes the peptide or aprecursor thereof). A precursor of the peptide can be particularlybeneficial where one or more of the amino acids are non-naturallyoccurring (e.g. a beta amino acid or an amino acid with a non-naturallyoccurring side chain). The manufacture of peptides chemically andrecombinantly are generally practiced in the art and are described in,for example, U.S. application Ser. Nos. 08/346,849 and 08/784,606 andAusubel, Current Protocols in Molecular Biology (1997). The peptides canpreferably be purified prior to use in the manufacture of the SAMs bystandard techniques, including HPLC.

The peptides employed in the invention are imprinted or patterned on asolid support. The shape of the solid support is not critical to theinvention and can be selected to optimize ease of use in the particularapplication. Thus, the solid support can be substantially spherical(e.g., a bead) or non-spherical, such as in a container (e.g., a petridish or cup), cylinder or cone, or a substantially flat film, stick,chip or disc, of essentially any size suitable for the ultimateapplication. The solid support can be porous (as in a membrane) ornon-porous (as in a petri dish or container).

The material employed in the manufacture of the solid support is notcritical as well. Thus, a variety of materials can be employed in themanufacture of the solid support. For example, the solid support can bean inorganic material such as a metal, including as gold, copper, zinc,silver or nickel or a metal alloy. Alternatively, the solid support canbe glass, silica, or silicon oxide. In yet another embodiment, the solidsupport can be an organic material, such as a polymer or resin,including nylon, poly(ethylene glycol), and polyfluoropolymers. It canbe desirable in some embodiments to employ a transparent solid support.In this embodiment, the detection of the binding of an opaque target(e.g., a cell) can be determined readily and accurately visually orelectronically and/or robotically employing, for example, a laser underthe control of a computer.

The solid support is selected with a view towards its ability to reactwith the terminal reactive group of the peptide. For example, the thiolgroup (e.g., X) can react with gold under standard methods, asdescribed, for example in Mrkisch and Whitesides, above. Likewise, thehydroxy group (e.g., X) can react with siloxane under relatively mildconditions. Xia, et al. “Microcontact Printing of Octadecylsiloxane onthe Surface of Silicon Dioxide and Its Application in Microfabrication,”J. Am. Chem. Soc. 117:9576-9577 (1995).

Solid supports which are inert to the peptide can be derivatized torender them reactive. For example, the solid support can be coated witha reactive material, chemically treated (e.g., by electrophilic ornucleophilic substitution reaction, addition reactions, etc.) tointroduce reactive groups.

The peptides are printed on the solid support, as will be describedbelow. The terms “printed”, “patterned” or “predetermined pattern” aredefined herein to mean that the solid support has ordered areas wherethe peptides are bonded and not bonded to the solid support. That is, aprinted or patterned solid support is expressly not intended to includea support with random or substantially homogeneous distribution of thepeptide over its entire surface(s). Furthermore, the peptides areprinted on the solid support in a single layer in a substantiallyconsistent configuration. Thus, the terms are further not intended toinclude solid supports wherein peptides are bonded to the solid supportvia distinct and different functional groups across the same molecule(e.g., distinct cysteine residues in a protein containing multiplecysteines along its sequence).

The patterns which can be selected in this invention are notparticularly critical. Preferred patterns for SAMs useful as researchtools in the study of cell/cell interactions are linear tracks ofalternating peptides which can adhere to the cells and inert tracks ofsolid support or an inert compound bound to the solid support. Dependingupon the thickness of the tracks, the orientation of the cell canfurther be manipulated. That is a thin track can result in theorientation of the cells linearly. FIG. 3 exemplifies suitable patterns.

As stated above, methods for the manufacture of SAMs are generally knownin the art. U.S. Pat. Nos. 5,620,850 and 5,512,131 and PCT PublishedApplication Nos.: WO97/07429 and WO96/29629 decribed suitable methodsfor manufacture. Additional examples include Deng, Li, Milan Mrksich andGeorge M. Whitesides, “Self-Assembled Monolayers of AlkanethiolatesPresenting Tri(propylene sulfoxide) Groups Resist the Adsorption ofProtein,” J. Am. Chem. Soc., 118(21):5136-5137 (1996); Chen, ChristopherS., Milan Mrksich, Sui Huang, George M. Whitesides, Donald E. Ingber,“Geometric Control of Cell Life and Death,” Science, 276:1425-1428(1997); López, Gabriel P., Mark W. Albers, Stuart L. Schreiber, ReedCarroll, Ernest Peralta, and George M. Whitesides, “Convenient Methodsfor Patterning the Adhesion of Mammalian Cells to Surfaces UsingSelf-Assembled Monolayers of Alkanethiolates on Gold,” J. Am. Chem.Soc., 115(13):5877-5878 (1993); Kumar, Amit, Nicholas L. Abbott, EnochKim, Hans A. Biebuyck, and George M. Whitesides, “PatternedSelf-Assembled Monolayers and Meso-Scale Phenomena,” Acc. Chem. Res.,28(5):219-226 (1995); DiMilla, Paul A., John P. Folkers, Hans A.Biebuyck, Ralph Härter, Gabriel P. López, and George M. Whitesides,“Wetting and Protein Adsorption of Self-Assembled Monolayers ofAlkanethiolates Supported on Transparent Films of Gold,” J. Am. Chem.Soc., 116(5):2225-2226 (1994); Singhvi, Rahul, Amit Kumar, Gabriel P.Lopez, Gregory N. Stephanopoulos, Daniel I. C. Wang, George M.Whitesides, Donald E. Ingber, “Engineering Cell Shape and Function,”Science, 264:696-698 (1994); Mrksich, Milan and George M. Whitesides,“Using Self-Assembled Monolayers to Understand the Interactions ofMan-Made Surfaces with Proteins and Cells,” Annu. Rev. Biophys. Biomol.Struct., 25:55-78 (1996); Wilbur, James L., Amit Kumar, Enoch Kim,George M. Whitesides, “Microfabrication by Microcontact Printing ofSelf-Assembled Monolayers,” Adv. Mater. 6(7/8):600-604 (1994); Xia,Younan, Enoch Kim, Milan Mrksich and George M. Whitesides, “MicrocontactPrinting of Alkanethiols on Copper and Its Application inMicrofabrication,” Chem. Mater. 8(3):601-603 (1996); Mrksich, Milan,Jocelyn R. Grunwell and George M. Whitesides, “Biospecific Adsorption ofCarbonic Anhydrase to Self-Assembled Monolayers of Alkanethiolates thatPresent Benzenesulfonamide Groups on Gold,” J. Am. Chem. Soc.,117(48):12009-12010 (1995); Jeon, Noo Li, Ralph G. Nuzzo, Younan Xia,Milan Mrksich, and George M. Whitesides, “Patterned Self-AssembledMonolayers Formed by Microcontact Printing Direct Selective Metalizationby Chemical Vapor Deposition on Planar and Nonplanar Substrates,”Langmuir, 11(8):3024-3026 (1995); Xia, Younan, Milan Mrksich, Enoch Kimand George M. Whitesides, “Microcontact Printing of Octadecylsiloxane onthe Surface of Silicon Dioxide and Its Application in Microfabrication,”J. Am. Chem. Soc., 117(37):9576-9577 (1995). The contents of thesearticles are incorporated herein by reference. The method is illustratedin FIGS. 1 and 2.

Referring specifically to FIG. 1, a polymeric or elastomeric stamp 1(e.g. a polydimethylsiloxane stamp) is contacted or “inked” with asolution 2 containing the peptide in a suitable solvent and then theinked stamp is pressed against the solid support 3, thereby transferringthe peptide solution in a controlled fashion to the solid support 3. Thepeptide is then maintained in contact with the solid support 3 underconditions suitable for binding, resulting in a SAM 4.

Upon binding of the peptide to the solid support, the solvent isgenerally removed, for example, by washing (e.g., extraction),evaporation or lyophilization.

The patterned SAM thus formed can then be used directly or can befurther derivatized, e.g., by subjecting the SAM to a second printingstep to ink a different chemical compound thereon. The second chemicalcompound can preferably be a peptide of the claimed invention or can bedifferent, such as an alkanethiol or poly(ethylene glycol), as describedin Mrksich and Whitesides, above.

In yet another alternative, the SAM can be subjected to additional stepswhich can modify the peptide on the SAM. This embodiment may bedesirable where the presenting group (e.g., L) or the chemical bond tothe central linker ((AA)_(m)) is labile under the conditions for bindingthe peptide to the solid support. Thus, the presenting group can bechemically reacted with a peptide precursor bonded directly to the solidsupport, thereby obtaining a SAM of the present invention.

In many instances, it can be desirable to modify the exposed areas ofthe solid support, for example, by exposing the SAM to ultraviolet lightor oxidize the SAM. This can be done to improve the reactivity oreliminate reactivity of the material of the solid support with one ormore materials encountered in storage or in use of the SAM.

Referring to FIG. 2, the solid support 3 is stamped with a solutioncontaining a first compound 5 (such as a poly(alkoxyglycothiol)) whichcan react with the solid support and presents an imprint or pattern ofthe solid support, as described above. The printed solid support 6 isthen contacted with a solution containing the peptide 2 under conditionssuitable for reacting the peptide with the exposed solid support. Thethus formed SAM 7 possess a pattern of the peptide in the relief of theimprint of the first compound. The SAM can then be washed and dried,. asabove. The printed solid support 6 can be immersed into a solution ofthe peptide or the peptide can be poured or sprayed onto the surface ofthe SAM, as is convenient.

Solvents which can be used to ink the peptide onto the stamp and, then,onto the solid support include solvents which can disperse or,preferably, solubilize the peptide. The solvent is preferably readilyremoved, for example, by evaporation, lyophilization or extraction, fromthe solid support. Examples of preferred solvents include alcohols, suchas ethanol, acetone, acetonitrile, DMSO and DMF and misciblecombinations thereof. The peptide solution concentration is selectedsuch that the desired amount of peptide is delivered to the solidsupport. That is, if it is desired to print the peptide upon the solidsupport at a high concentration or density, then the peptide solutioncan be at or near the saturation level of a good solvent. If it isdesired to imprint a low concentration of the peptide sparsely upon thesolid support, the solution can be characterized by a low concentrationsuch as employing a dilute solution.

The solution comprising the peptide can also include additionalcomponents. For example, a dispersant or solubilizer can be added to thesolution to solubilize or disperse, for example, the peptide. It can bedesirable in some instances to include a colorant in the solution,particularly where the solution is colorless or is difficult to observeon the solid support or stamp, so that the area of the solid supportwhich has been inked can be visually observed. It is generally desirablewhere additional components are added to the solution that they can bereadily removed from, e.g. washed free of, the solid support.

It is clear that, in the method for manufacturing the SAMs, either thestamp, solid support or both can be mobile, relative to the other. Thatis, the stamp can be fixed and the solid support pressed firmly againstit or vice versa. Alternatively, both the stamp and support can bemobilized. This process can be readily achieved employing robotics,which ensures a high degree of consistency and accuracy in the printingstep.

The peptide can be bound to the solid support via covalent bonding,ionic bonding or other chemical interactions. It is preferred that thebonding be of a high affinity and be essentially irreversible under theconditions for use. The conditions suitable for bonding the peptide tothe solid support can be dependent upon the nature of the chemicalreaction relied upon and can generally be determined by the person ofskill employing no more than routine skill.

Clearly, other methods for the manufacture of the SAMs of the presentinvention will be apparent to the person of skill in the art and areintended to be included within the scope of the present invention.

The SAMs of the invention can be employed in a variety of processes inbiology, biotechnology, medicine, material science, biomedicalengineering and computer-related inventions. A preferred example of anapplication includes the use of the SAMs as substrates for ELISA.

SAMs to Screen for the Presence of a Target in a Sample

The SAMs of the present invention can be used to screen for the presenceof a target in a sample. As set forth above, the SAMs of the inventioncan be designed to possess a presenting group which binds specificallyor non-specifically to a target or target molecule. Where the presenceof a cell is to be detected and distinguished from other cells in thesample (e.g.,the presence of a tumor cell in a tissue sample which canfurther comprise normal diploid cells), the presenting group is“specific” to the target, i.e. does not bind substantially to othermaterials or cells which can be present. Where the presence of manydifferent cells in a sample (e.g., the presence of bacterialcontaminants in a pharmaceutical process stream), the presenting groupis non-specific to a particular target but can bind to a large number oftargets.

The method of screening for the presence of a target can comprise thesteps of contacting an SAM, as described above, with a sample underconditions suitable for the target or target molecule to bind to thepresenting group on the SAM and detecting the presence of the target ortarget molecule. The target or target molecule can be a cell, such as amammalian cell (e.g.,tumor cell, normal diploid somatic cell, or stemcell), a bacterium or yeast (e.g., a causative agent for disease orcontaminant). Alternatively, the target or target molecule can be avirus (e.g., a causative agent for disease or contaminant), toxin orprotein, etc.

The sample can be obtained from an animal or patient, such as a tissuesample or biopsy, body fluid, e.g., serum, milk, saliva or urine orfecal matter. Alternatively, the sample can be obtained frommanufacturing process, such as a pharmaceutical process or food process.Thus, the method can be used to screen for contaminants or sterileconditions in manufacturing or it can be used to screen for or diagnosedisease in a patient.

It is generally desirable that the sample be contacted with the SAM as aliquid, e.g. a dispersion or solution. Thus, the sample can be mixedwith a diluent or buffer. Examples of diluents include water, such assterile water, polar and non-polar solvents, e.g. alcohols,dimethylformamide, acetonitrile, alkanes, benzene, toluene, etc. Buffersinclude physiological buffers, such as phosphate buffered solution,culture media, etc.

The person of skill in the art can determine empirically the conditionsfor contacting the SAM and the sample such that the target or targetmolecule can react with each other and bind. Such conditions are wellknown in the art. Generally, where the method is a diagnostic tool andthe sample is a tissue sample or other biological sample, the conditionswill physiologic. That is, physiological pH is generally employed. Roomtemperature can also be employed in many instances. Where the method isdetecting the presence of contaminants in a sample, neutral pH can begenerally employed, as well as room temperature.

The SAM can be contacted with the sample in a number of ways. Forexample, the SAM can be immersed into the sample, as in dipping a stick.Alternatively, the sample can be poured over or through the SAM.Optionally, the SAM can be rinsed after the contacting step, such aswith sterile water.

After the SAM has been contacted with the sample, the presence of thetarget or target molecule is detected. This can also be performed in anumber of ways. In one embodiment, the SAM can be contacted with asecond solution which possesses a labeled compound which can react withthe target molecule, as in an ELISA method. The label (e.g., acolorimetric label or radiolabel) can then be detected. In manyembodiments, the target can be detected visually, with the naked eye,under a microscope or robotically. This can be advantageous, forexample, where the target is a cell. In many embodiments, it may bedesirable to permit any cells bound to the SAM to colonize prior todetection. The method of the invention can accurately determine thepresence of an individual cell or determine a precise cell count in asample.

In a particularly preferred method, the solid support for the SAM istransparent. In such an embodiment, the presence of an opaque target,such as a cell, can be determined by scanning the SAM with a laser anddetermining the number of targets or cells present thereon, whichaccurately correlates to the number of interruptions in scanning. Thismethod can be performed in an automated system (e.g. robotically),thereby improving efficiency and avoiding inaccurate results due tohuman error.

SAMs in Cell Culture

The SAMs of the invention can be used as a solid support in culturingcells. Cells can be attached to the SAMs by contacting the cells to beattached with the SAM and maintaining the cells under conditionssuitable for growth. As above, it is generally desirable to contact thecells with the SAM as a liquid, e.g., in the presence of a diluent orsolvent. The cells can be attached to the solid support in apredetermined fashion, order and orientation.

Conditions for maintaining cells can be those employed routinely for thecell or cell type to be cultures. For example, the culture can bemaintained under temperatures (e.g. between about 25° C. to about 60°C.) and pH (e.g. between about 4 and about 10) appropriate for growth.Nutrients appropriate for growth can also advantageously be provided tothe culture.

The invention permits very accurate control of cell population anddensity. The invention can be utilized to study cell growth and cellularinteractions to external stimuli, including other cells, growth factors,repellants and inhibitors. Thus, the invention represents a significantadvance in the ability to conduct research in biology and medicine.

In yet another embodiment, the method can be employed in screenings orassays employing cells, such as screening for drugs which may inhibitthe growth of a cell or cells (such as in a screen for anti-tumoragents, anti-bacterials). Alternatively, the method can be employed inthe screening for drugs which increase or activate the growth of a cellor cells, including fibroblasts, endothelial cells, smooth muscle cells,hematopoietic cells and neuronal cells, etc.

The method can also be used to maintain cell cultures, including tissuecultures, in the manufacture of cellular products (e.g., proteins,hormones, etc.), artificial tissues, etc. Examples of tissues which canbe cultured in this manner include fibroblasts, endothelial cells,smooth muscle cells and neuronal cells. Such tissues can be employed asgrafts, such as autologous grafts.

The understanding of complex neuronal connections is central to ourcomprehension of central nervous system function, and advances in doingso will benefit from combining engineering with molecular cell biologyto analyze neuronal behavior under well-characterized and controlledconditions. Neurite outgrowth, guidance and connections can be studiedon surfaces patterned with self-assembling peptides that containcell-adhesion motifs. Controlling neurite outgrowth, includingdistances, angles and direction, can be important in controlling andstudying synapse formation between neuronal cells guided into proximity.Neuronal cells attached to the described SAMs can be employed in thestudy of neuronal cell culture, synapse formation, neuronal connectionengineering, screening neuropeptides, as well as pharmaceutical agentsthat stimulate, inhibit or alter the nature of nerve growth, andinter-connections. For example, attractants, e.g., growth factors,neuropeptides, neurotrophins, and drugs can be screened for theirability to alter the direction or growth behavior of neurites or theirability to induce, stimulate, suppress or inhibit neurite growth. Theseattractants can be placed or randomly contacted with the neuronalcell-bound SAMs.

Preferred peptides for the manufacture of the SAMs for this applicationinclude peptides wherein the presenting group is a cell adhesion motifor peptide which binds to neuronal cells. Examples of suitable celladhesion motifs are (RADX)_(n), (RADS)_(n), (EAKX)_(n), and (EAKS)_(n),wherein X is an amino acid, such as S, and n is an integer, preferablybetween about 2 to about 8. Oligopeptides of these sequences have beenshown to promote neurite outgrowth in culture (U.S. application Ser. No.08/784,606, which is incorporated herein by reference in its entirety).

EXAMPLE 1 Preparation of Patterned SAMs Glass Chip

A 10:1 (w:w) mixture of Sylgard Silicone Elastomer 184 and SylgardCuring Agent 184 (Dow Corning Corp., Midland, Mich.) was casted over amaster, which was generated by photolithography, and pressure degassed.After sitting at room temperature for 1 hour, the PDMS was cured at 60°C. for 2 hours. The stamp was carefully peeled off the master aftercooling to room temperature and rinsed with ethanol. The PDMS stamp wasinked by a cotton swab which has been moistened with a 5 mM solution of(1-mercaptoundec-11-yl)hexa(ethylene glycol) (HO(CH₂CH₂O)₆(CH₂)₁₁SH) inethanol. The resulting stamp was placed on the gold substrate (125 ★gold on a titanium-primed 24×50-2 microscope cover glass) and gentlehand pressure was applied to aid in complete contact between the stampand the glass chip. After 1 minute, the stamp was peeled off the glasschip and the resulting substrate was immersed directly in a 2 mMsolution of (RADC)₃ AAAC (SEQ ID NO: 1) in distilled, deionized water.After approximately 2 hours of immesion, the glass chip was removed fromthe solution, rinsed extensively with water and ethanol, and dried witha stream of filtered nitrogen gas.

In our preliminary experiments, when the cells (of various types) areplated on surfaces coated with hexa-ethyleneglycolthiol, (EG)6-SH, theyrarely attach to the surface. In contast, cells attached very well whenplated on the surface coated with the “RADSC” peptide. In theseexperiments, after cell attachment, the plates containing cells weeshacked at 150 rpm for 10 minutes and the coated cover-slides werewashed in new medium and transferred to new plates in order to eliminateunattached cells.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A device comprising: a solid support; and an array of isolated regions on the support, the array comprising a layer of peptides, wherein the peptides are bound to the support by a bond between the support and a terminal amino acid in a preselected, reproducible pattern.
 2. The device of claim 1 further comprising a background region surrounding the isolated regions, wherein the peptides are not bound to the background region.
 3. The device of claim 2 wherein the background region comprises an inert compound.
 4. A device comprising: a solid support; and an isolated region comprising a layer of peptides, wherein the peptides are bound to the support by a bond between the support and a terminal reactive group in a preselected, reproducible pattern.
 5. The device of claim 4 comprising a plurality of isolated regions of a self-assembled monolayer, the plurality of regions defining an ordered array on the support.
 6. A device comprising: a solid support; an isolated region comprising a self-assembled monolayer of peptides, wherein the peptides are bound to the support by a bond between the support and a terminal reactive group in a preselected, reproducible pattern; and a background region surrounding the isolated region and comprising a compound which can react with the support.
 7. A device for immobilizing at least one biological material in a specific and predetermined pattern comprising: a surface, an array of immobilization islands in a specific and predetermined pattern over the surface isolated from each other by at least one background region, the array of immobilization islands comprising a first self-assembled monolayer comprising at least one first functional group wherein the at least one first functional group is selected to biophilic, and wherein the first self-assembled monolayer comprises a monolayer of linear peptides, the at least one background region comprising a second self-assembled monolayer having a second functional group wherein the second functional group is selected to be biophobic. 